Influenza is responsible for much morbidity and mortality in the world and is considered by many as belonging to the most significant viral threats to humans. Annual Influenza epidemics swipe the globe and occasional new virulent strains cause pandemics of great destructive power. At present the primary means of controlling Influenza virus epidemics is vaccination. However, mutant Influenza viruses are rapidly generated which escape the effects of vaccination. In the light of the fact that it takes approximately 6 months to generate a new Influenza vaccine, alternative therapeutic means, i.e., antiviral medication, are required especially as the first line of defense against a rapidly spreading pandemic.
An excellent starting point for the development of antiviral medication is structural data of essential viral proteins. Thus, the crystal structure determination of the Influenza virus surface antigen neuraminidase (von Itzstein et al., 1993) led directly to the development of neuraminidase inhibitors with anti-viral activity preventing the release of virus from the cells, however, not the virus production. These and their derivatives have subsequently developed into the anti-Influenza drugs, zanamivir (Glaxo) and oseltamivir (Roche), which are currently being stockpiled by many countries as a first line of defense against an eventual pandemic. However, these medicaments provide only a reduction in the duration of the clinical disease. Alternatively, other anti-Influenza compounds such as amantadine and rimantadine target an ion channel protein, i.e., the M2 protein, in the viral membrane interfering with the uncoating of the virus inside the cell. However, they have not been extensively used due to their side effects and the rapid development of resistant virus mutants (Magden et al., 2005). In addition, more unspecific viral drugs, such as ribavirin, have been shown to work for treatment of Influenza infections (Eriksson et al., 1977). However, ribavirin is only approved in a few countries, probably due to severe side effects (Furuta et al., 2005). Clearly, new antiviral compounds are needed, preferably directed against different targets.
Influenza virus as well as Thogotovirus belong to the family of Orthomyxoviridae which, as well as the family of the Bunyaviridae, including the Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus, are negative stranded RNA viruses. Their genome is segmented and comes in ribonucleoprotein particles that include the RNA dependent RNA polymerase which carries out (i) the initial copying of the single-stranded virion RNA (vRNA) into viral mRNAs and (ii) the vRNA replication. For the generation of viral mRNA the polymerase makes use of the so called “cap-snatching” mechanism (Plotch et al., 1981; Kukkonen et al., 2005; Leahy et al., 1997; Noah and Krug, 2005). It binds to the 5′ RNA cap of cellular mRNA molecules and cleaves the RNA cap together with a stretch of nucleotides. The capped RNA fragments serve as primers for the synthesis of viral mRNA. The polymerase is composed of three subunits: PB1 (polymerase basic protein), PB2, and PA. While PB1 harbors the endonuclease and polymerase activities, PB2 contains the RNA cap binding domain.
The polymerase complex seems to be an appropriate antiviral drug target since it is essential for synthesis of viral mRNA and viral replication and contains several functional active sites likely to be significantly different from those found in host cell proteins (Magden et al., 2005). Thus, for example, there have been attempts to interfere with the assembly of polymerase subunits by a 25-amino-acid peptide resembling the PA-binding domain within PB1 (Ghanem et al., 2007). Furthermore, the endonuclease activity of the polymerase has been targeted and a series of 4-substituted 2,4-dioxobutanoic acid compounds has been identified as selective inhibitors of this activity in Influenza viruses (Tomassini et al., 1994). In addition, flutimide, a substituted 2,6-diketopiperazine, identified in extracts of Delitschia confertaspora, a fungal species, has been shown to inhibit the endonuclease of Influenza virus (Tomassini et al., 1996). Moreover, there have been attempts to interfere with viral transcription by nucleoside analogs, such as 2′-deoxy-2′-fluoroguanosine (Tisdale et al., 1995) and it has been shown that T-705, a substituted pyrazine compound may function as a specific inhibitor of Influenza virus RNA polymerase (Furuta et al., 2005). Finally, by comparison studies between the binding mode of human cap binding protein eIF4E to RNA cap structures and Influenza virus RNP interaction with RNA cap structures Hooker et al. (2003) identified a novel cap analog that selectively interacts with Influenza virus, but not human cap binding protein. However, the major obstacle for identifying compounds that interact with the RNA cap binding pocket of PB2 and potentially interfere with RNA cap binding and thereby RNA polymerase activity was up to now that the structure and identity of said binding pocket was unknown.
Several attempts have been made to elucidate the RNA cap binding site, however, with controversial results. Cross-linking experiments indicated that two separate sequences, one N-(242-282) and one C-terminal (538-577) proximal segment of PB2, constitute the RNA cap-binding site of the Influenza virus RNA polymerase (Honda et al., 1999). Additional cross-linking experiments identified a sequence extending from amino acid 533 to amino acid 564 in the PB2 protein subunit, particularly amino acid residue Trp552, as potential interaction site for the RNA cap (Li et al., 2001). Furthermore, mutational analysis resulted in potential RNA cap binding amino acid residues Phe363 and Phe404 within PB2 (Fechter et al., 2003).
It is an object of the present invention to provide (i) high resolution structural data of the RNA cap binding pocket of PB2 by X-ray crystallography, (ii) computational as well as in vitro methods, preferably in a high-throughput setting, for identifying compounds that can bind to the RNA binding pocket of PB2, preferably competing with RNA cap binding and thereby interfering with RNA polymerase activity, and (iii) pharmacological compositions comprising such compounds for the treatment of infectious diseases caused by viruses using the cap snatching mechanism for synthesis of viral mRNA.
The present invention allows for the first time for the precise definition of the PB2 RNA cap-binding site within an independently folded domain. It has up to now been highly controversial where the site may be located. It was a common believe that a functional cap binding site requires all three polymerase subunits and possibly also viral RNA (Cianci et al., 1995; Li et al., 2001). The surprising achievement of the present inventors to recombinantly produce soluble PB2 polypeptide fragments comprising a functional RNA cap binding pocket allows to perform in vitro high-throughput screening for inhibitors of a functional site on Influenza virus polymerase using easily obtainable material from a straightforward expression system. Previous work on cap binding inhibitors has, for instance, used complete ribonucleoprotein particles purified from Influenza virions (Hooker et al., 2003). Furthermore, by providing detailed structure coordinates of the RNA cap binding pocket within PB2, the present invention allows to use structure-based approaches to cap-binding inhibitor design, i.e., in silico screening and lead optimization.